Methods and related compositions using specific flavonoids and indanes to reduce weight and inhibit lipase, α-amylase and α-glucosidase activity in mammals

ABSTRACT

The present invention relates generally to methods and related compositions using flavonoids and/or indanes extracted from the stems and leaves of  C. quadrangularis  to reduce weight and inhibit lipase, α-amylase and α-glucosidase activity in mammals. By example and not by way of limitation, embodiments of the present disclosure, a composition and related methods for reducing body weight and/or inhibiting any combination of lipase, α-amylase and α-glucosidase is provided. The composition contains an effective amount of one or more flavonoids or indanes selected from 3-O-rhamnopyranosylkaempferol, 3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol, quercitrin, rhamnitrin, rhamnocitrin, quercitrin-3-O″-acetate and parthenocissin A.

RELATED APPLICATION

This application is a divisional of U.S. Application Ser. No.11/855,084, filed Sep. 13, 2007, the contents of which are incorporatedby reference herein in its entirety.

BACKGROUND

Cissus quadrangularis L. (Vitaceae) is an ancient medicinal plant nativeto the hotter parts of Ceylon and India. The stems of Cissusquadrangularis L (Vitaceae) have been consumed for centuries throughoutAsia and Africa as a culinary vegetable. C. quadrangularis has been usedin India for promoting the fracture healing process [1] for some time.It has been prescribed in Ayurveda as an anthelmintic, dyspeptic,digestive tonic, analgesic in eye and ear diseases and in the treatmentof irregular menstruation and asthma. In Cameroon, the whole plant isused in oral re-hydration, while the leaf, stem and root extracts ofthis plant are important in the management of various ailments.Accordingly, the safely of C. quadrangularis, without causingundesirable side effects, has been demonstrated over time.

There are several reports on C. quadrangularis use in the management ofobesity and complications associated with metabolic syndrome [2], andits antioxidant and free radical scavenging activity in vitro [3,4].Various formulations now contain extracts of C. quadrangularis incombination with other compounds, used for the purpose of weightmanagement and complications/conditions resulting from these and relatedconditions.

Although the use of C. quadrangularis has been shown to provide certainbenefits and advantages, it is unclear what components or aspects of C.quadrangularis provide what desirable benefit and advantages.Additionally, it is unclear whether certain components or aspects of C.quadrangularis counteract the benefits or efficacy of other componentsor aspects of C. quadrangularis. Furthermore, novel compositions andrelated methods which safely and effectively provide improved weightloss in a mammal, without causing undesirable side effects, is desired.Additionally, compositions and related method that safely andeffectively inhibits lipase, α-amylase and α-glucosidase activity in amammal, thereby contributing to weight loss and overall health, withoutcausing undesirable side effects, is desired. The compositions andrelated methods of the present invention provide these and other relatedbenefits and advantages.

SUMMARY OF THE INVENTION

The present invention relates to methods and related compositions usingflavonoids and/or indanes extracted from the stems and leaves of C.quadrangularis to reduce weight and inhibit lipase, α-amylase andα-glucosidase activity in mammals. By example and not by way oflimitation, in one embodiment of the present disclosure, a compositionfor reducing body weight or inhibiting any combination of lipase,α-amylase and α-glucosidase is provided. The composition contains aneffective amount of flavonoid 3-O-rhamnopyranosylkaempferol or indane3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diolor flavonoid 3-O-rhamnopyranosylkaempferol and indane3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol.

In another embodiment of the present disclosure, a method for reducingbody weight in a mammal is provided. The method including providing acomposition containing an effective amount of one or more flavonoids orindanes selected from 3-O-rhamnopyranosylkaempferol,3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol,quercitrin, rhamnitrin, rhamnocitrin, quercitrin-3-O″-acetate andparthenocissin A and claiming that the composition reduces, maintains orimproves body weight in a mammal.

In yet another embodiment of the present disclosure, a method forinhibiting any combination of lipase, α-amylase and α-glucosidase in amammal is provided. The method including providing a compositioncontaining an effective amount of one or more flavonoids or indanesselected from 3-O-rhamnopyranosylkaempferol,3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol,quercitrin, rhamnitrin, rhamnocitrin, quercitrin-3-O″-acetate andparthenocissin A and claiming that the composition inhibits anycombination of lipase, α-amylase and α-glucosidase in a mammal. In adetailed aspect of the present disclosure, the effective amount of thecomposition provided to the mammal is 10 mg to 900 mg daily. In anotherdetailed aspect of the present disclosure, the effective amount of thecomposition is 50 mg to 200 mg daily.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 demonstrates the seven flavonoid and indanes that were tested fortheir ability to inhibit lipase, α-amylase and α-glucosidase.

FIG. 2 is a chart summarizing the experimental results, in % inhibition,of the enzyme inhibition activity of the seven flavonoids and indaneslisted in FIG. 1. Samples were tested at 0.15 mg/ml (lipase), 0.12 mg/ml(amylase) and 0.28 mg/ml (glucosidase) and the results are mean values(n=3).

DETAILED DESCRIPTION

Phytochemical analyses of C. quadrangularis have revealed high contentsof ascorbic acid, carotene, anabolic steroidal substances and calcium.The stem contains two asymmetric tetracyclic triterpenoids, and twosteroidal principles [5]. The presence of β-sitosterol, δ-amyrin andδ-amyrone has also been reported. All of these components havepotentially different metabolic and physiological effects. Throughexperiments we performed, two novel components of C. quadrangularis,flavonoid (3-O-rhamnopyranosylkaempferol) and indane(3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol),demonstrated inhibition of enzymes known to effect weight loss inmammals.

More specifically, our experiments, which are discussed in detail below,have shown that aqueous extracts of C. quadrangularis stems and leavescontains specific flavonoids and/or indanes which inhibit lipase,amylase and/or α-glucosidase and, therefore, are least in part relatedto the anti-obesity activity of the plant. The enzyme inhibitionactivity of a novel flavonoid (3-O-rhamnopyranosylkaempferol) and indane(3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol)(7) and four known structurally related flavonoids and one indaneisolated from the extract were tested.

Preliminary extraction and bioassays had shown that aqueous extracts ofC. quadrangularis stems/leaves inhibited pig amylase, human lipase andyeast α-glucosidase. The inhibition of these enzymes was shown to beselective by comparison with the lack of activity against a panel ofother commercially (Sigma) available enzymes including β-glucosidase(almond), α-(green coffee beans) and β-galactosidase (A. niger) andα-fucosidase (human). Bioassay guided fractionation of the extract wasperformed using the assays for amylase, lipase and α-glucosidase. Theaqueous plant extract was passed down a cation exchange resin (Dowex 50H+ form) from which the inhibitors were un-retained. The extract wasapplied to a HP20 resin and three active fractions obtained; unbound,25% methanol elution and 10% acetone in methanol elution.

All showed enzyme inhibition and similar chemical profiles but the 10%acetone in methanol fraction was more suitable for compoundpurification. Further chromatography of this fraction by polar flashchromatography on silica and preparatory and semi-preparatory HPLC onC18 gave seven compounds shown in FIG. 1. Five have been previouslyreported; quercitrin (1), rhamnitrin (3) and rhamnocitrin (4),quercitrin-3-O″-acetate (5) and parthenocissin A (6) and two were novel,3-O-rhamnopyranosylkaempferol (2) and3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol(7).

Inhibition of Enzyme Activity

As shown in FIG. 2, all but one of the flavonoids show good α-amylaseinhibition. Quercitrin (1) differs from the novel3-O-rhamnopyranosylkaempferol (2) only by one hydroxyl substituent butthis results in a decrease in α-amylase inhibition. However, methylationof a ring hydroxyl, in the case of rhamnitrin (3), or acetylation of thesugar, in the case of quercitrin-3-O″-acetate (5) causes the inhibitionto be restored. All the flavonoids show similar response to lipaseinhibition but again the inhibition of α-glucosidase shows variation forthe various structural features.

The two indanes, 6 and 7 display significantly different inhibitionprofiles. Parthenocissin A (6) shows good inhibition of α-amylasewhereas the novel3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol(7) is fairly specific as an inhibitor of the α-glucosidase.

Our experiments did not determine the K_(i) values of the more potent ofthe individual inhibitors since we were more interested in indicatingthe combined effect of multiple inhibitors taken together as found forthe original extract. It is important in such studies, however, to lookfor some specificity of inhibition to prove that the inhibition is not atherapeutically unattractive non-specific interaction with proteins ingeneral. We have shown here that the compounds show specific inhibitionof different enzymes. The inhibition of lipase did not go above 60% evenwith use of the commercial inhibitor Orlistat® at the same topconcentration. The lipase and amylase seemed much more prone toinhibition by the C. quadrangularis components in general than theα-glucosidase.

The presence of several components exhibiting inhibition of amylase,lipase and glucosidase suggests that the plant could well have an effecton the ability of the gastrointestinal tract to utilize the plant andother sources of nutrients efficiently. Inhibitors of lipase such asOrlistat® and glucosidases such as Glyset® and Acarbose® are used asdrugs to treat obesity and diabetes type 2. These drugs, however, oftencause side-effects and it may well be that the combination of severaltested components in C. quadrangularis working on different enzymes hasa better tolerated effect and reduces or eliminates side effectsassociated with commercially available pharmaceuticals.

Although the combined effect of the aqueous extract was inhibition ofthe glucosidase activity, it is of interest to note that 1 and 5actually promoted the glucosidase activity. Such promotion of enzymeactivity can be due to binding to allosteric sites on the enzyme. Whilethe effect of increased glucosidase activity by some components onweight control or diabetes is difficult to understand, there are manyα-glucosidases present in the gastrointestinal tract and within cellsand inhibition of some may be beneficial to the control of weight ordiabetes whereas increased activity of certain glucosidases may bepromoting health in other ways. We have only used one α-glucosidase hereand the compounds may vary in effects and activity on otherα-glucosidases. The ability of the individual components identified toreach the sites of enzyme activity will also vary and will be influencedby individual differences and no doubt also by variations in other foodconsumed with C. quadrangularis and formulation of products containingit.

The inhibition of α-glucosidases has been shown to be potentiallytherapeutically useful in diseases other than diabetes type 2 and weightcontrol [6]. In particular such inhibitors have been studied aspotential anti-viral and anti-cancer agents and it may well be thatinhibition of α-glucosidases by C. quadrangularis explains the widetraditional medicinal use of the species. Promotion of specificglycosidase activities can also be therapeutic where those enzymes aredeficient [6].

Experimental

Structure Elucidation

Structure elucidation was carried out using NMR (Bruker DRX500) for full¹H and ¹³C assignments and LCMS (Waters Integrity), which gave EIMS datafor molecular weight and/or fragmentation assignment. All of thecompounds would require further work (optical rotation measurementsand/or x-ray crystallography) to ascertain stereochemistry.

Literature searches were conducted using the Dictionary of NaturalProducts (DNP) available on CDROM.

Plant Material

Cissus quadrangularis was supplied dry from the Cameroon. The referencefor the voucher specimen for Cissus quadrangularis is No. 18668/SRF/Cam,identified by the Cameroon National Herbarium, Yaounde, Cameroon.

Extraction and Isolation

The dried Cissus quadrangularis (2.3 kg) was extracted in 50% aqueousethanol (10 L) overnight. The filtered extract was loaded on two cationexchange columns (Dowex 50 H⁺ form, 700 cm³) from which the inhibitorswere un-retained. The extract (8 L) was applied to a HP20 cartridge(6×75 cm) pre-equilibrated with 50% aqueous methanol (5 L) and elutedwith 25% aqueous methanol (3 L) followed by 10% acetone in methanol (6L) to give three active fractions; unbound (37 g), 25% methanol elution(27 g) and 10% acetone in methanol elution (14 g). All showed enzymeinhibition and similar chemical profiles but the 10% acetone in methanolfraction was more suitable for compound purification. The 10% acetone inmethanol extract was bound onto silica and applied to a KP-Sil™ silicaFlash 75S cartridge (7.5×9.0 cm) pre-equilibrated with heptane (5 L) andeluted with 75% heptane in ethyl acetate (1 L), 50% heptane in ethylacetate (1 L), 25% heptane in ethyl acetate (1 L), 100% ethyl acetate (1L) and 95% ethyl acetate in methanol (1 L) to give 5 fractions.

Preparatory HPLC of fraction 4 on Water's Nova-Pak® HR C18 column(2×(40×100 mm) in series, 6 μm, 60 A⁰ at a flow rate of 55 ml/min andmonitoring wavelength of 225 nm with an acetonitrile/water gradient; 80%water:20% acetonitrile containing 0.1% TFA to 65% water:35% acetonitrilecontaining 0.1% TFA over 15 mins, gave rise to parthenocissin A (6).Further purification of this fraction on Water's Nova-Pak® HR C18 column(25×100 mm, 6 μm, 60 A⁰ at a flow rate of 15 ml/min and monitoringwavelength of 225 nm with an methanol/water gradient; 55% water:45%methanol containing 0.1% TFA to 30% water:70% methanol containing 0.1%TFA over 15 mins, gave rise to quercitrin-3-O″-acetate (5) and3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyphenyl)-1-(4-hydroxyphenyl)indane-4,6-diol(7). Preparatory HPLC of fraction 5 on Water's Nova-Pak® HR C18 column(2×(40×100 mm) in series, 6 μm, 60 A⁰ at a flow rate of 55 ml/min andmonitoring wavelength of 210 nm with an acetonitrile/water gradient, 80%water:20% acetonitrile containing 0.1% TFA to 60% water:40% acetonitrilecontaining 0.1% TFA over 20 mins, gave rise to quercitrin (1),3-O-rhamnospyranosylkaempferol (2), rhamnitrin (3) and rhamnocitrin (4).

Kits and Reagents Used for Bioassays

The purified compounds and extracts were made up 2 mg/ml solutions inwater using initially a drop of DMSO if required for solubility.

Lipase

Sigma Lipase-PS Kit (Catalogue Number 805-A)

Human pancreatic lipase was diluted 1.5:5 prior to use. The assay wascarried out at 20° C. using 10 μl test compound, 5 μl enzyme solution,90 μl substrate, and 30 μl activator reagent. Color formation wasmeasured at 550 nm after 30 minutes.

Amylase

Sigma INFINITY Powder Reagent (Catalogue Number 568-20)

Sigma α-amylase (A6255, porcine pancreatic amylase as salinesuspension), diluted to 35 units/ml. The assay was carried out at 20° C.using 10 μl enzyme solution, 10 μl test compound, and 150 μl INFINITYreagent. The rate of color formation was measured over five minutes,after which the final absorbance was measured at 405 nm as an endpointrecording.

α-Glucosidase

Sigma α-glucosidase (G5003, from S. cerevisiae) at 2 units/ml inphosphate buffer pH 6.0. Sigma p-nitrophenyl-α-D-glucopyranoside, 5 mMin phosphate buffer pH 6.0. The assay was carried out at 20° C. using 10μl enzyme solution, 10 μl test compound, and 50 μl substrate solution.The reaction was carried out for 7 minutes; color formation was measuredat 415 nm following the addition of 80 μl 0.4 M glycine solution, pH10.4 to stop the reaction. Other glycosidase assays were carried outusing enzymes from Sigma and appropriate p-nitrophenyl substrates [7].

3-O-rhamnopyranosylkaempferol (2)

UV/Vis λ_(max) (MeOH) nm (log ε): 200, 264, 342 ¹H NMR (500 MHz, MeOH) δppm 0.79 (3H, d, J=6 Hz), 3.23 (1 H, m), 3.59 (1 H, m), 3.59 (1 H, m),4.11 (1 H, m), 5.27 (1H, d, J=2 Hz), 6.10 (1H, d, J=2 Hz), 6.27 (1H, d,J=2 Hz), 6.84 (2H, dd, J=2.9 Hz), 7.67 (2H, dd, J=2.9 Hz). ¹³C NMR (500MHz, MeOH) δ ppm 18.0 (CH₃), 72.3 (CH), 72.4 (CH), 72.6 (CH), 73.6 (CH),95.2 (CH), 100.3 (CH), 103.9 (CH), 106.4 (C), 117.0 (2×CH), 123.1 (C),132.3 (2×CH), 136.7 (C), 159.0 (C), 159.7 (C), 162.0 (C), 163.7 (C),166.3 (C), 180.1 (C═O). HPLC-MS: m/z, 288 (100) (C₂₁H₂₂O₁₀), 257, 120.

3-(4-hydroxybenzylidene)-2-(2,5-dihydroxy-phenyl)-1-(4-hydroxyphenyl)indane-4,6-diol(7)

UV/Vis λ_(max) (MeOH) nm (log ε): 200, 323 ¹H NMR (500 MHz, MeOH) δ ppm4.21 (1H, broad s), 4.36 (1H, broad s), 6.27 (1H, t, J=2 Hz), 6.35 (1H,d, J=2 Hz), 6.41 (2H, d, J=2 Hz), 6.81 (4H, dd, J=2.9 Hz), 6.88 (1 H, d,J=2 Hz), 7.07 (2H, dd, J=2.9 Hz), 7.16 (1H, s), 7.29 (2H, d, J=9 Hz).¹³C NMR (500 MHz, MeOH) δ ppm 58.5 (CH), 61.6 (CH), 98.9 (CH), 102.0(CH), 104.3 (CH), 107.1 (2×CH), 116.3 (2×CH), 116.4 (2×CH), 123.6 (CH),125.9 (C), 129.3 (2×CH), 129.6 (2×CH), 130.7 (C), 131.6 (CH), 139.0 (C),143.8 (C), 148.0 (C), 148.1 (C), 150.1 (C), 156.5 (C), 156.9 (C), 157.8(C), 160.1 (2×C). HPLC-MS: M·⁺ 454 (C₂₈H₂₂O₆, m/z 360, 347 (100), 331,239, 227, 226, 215, 199, 180, 149, 107, 95.

REFERENCES

-   [1] Chopra S, Patel M, Gupta L and Datta I. (1975) Studies on C.    quadrangularis in experimental fracture repair: Effect on chemical    parameters in blood. Indian Journal of Medicinal Research, 63:    824-828.-   [2] Oben J, Kuate D, Agbor G, Momo C and Talla X. (2006) The use of    a C. quadrangularis formulation in the management of weight loss and    metabolic syndrome. Lipids in Health and Disease, 5; 24-30.-   [3] Agbor G, Kuate D, Oben J. (2007) Medicinal plants can be a good    source of antioxidant: Case study of Cameroon, Pakistan Journal of    Biological Science, 10, 537-544.-   [4] Mallika J and Shyamala C. (2005) In vitro and in vivo evaluation    of free radical scavenging potential of C. quadrangularis. African    Journal of Biomedical Research., 8: 95-99.-   [5] Mehta M, Kaur N, Bhutani K K. (2007) Determination of marker    constituents from Cissus quadrangularis Linn. and their quantization    by HPTLC and HPLC. Phytochemical Analysis 12, 91-105.-   [6] Watson A, Fleet G W J, Asano N, Molyneux R J and Nash R J (2001)    Polyhydroxylated Alkaloids—Natural Occurrence and Therapeutic    Applications. Phytochemistry 56, 265-295.-   [7] Watson A, Nash R, Wormald M, Harvey D, Dealler S, Lees E, Asano    N, Kizu H, Kato A, Griffiths R C, Cairns A J and Fleet G W J. (1997)    Glycosidase-inhibiting pyrrolidine alkaloids from Hyacinthoides    non-scripta. Phytochemistry 46, 255-259.

While the apparatus and method have been described in terms of what arepresently considered to be the most practical and preferred embodiments,it is to be understood that the disclosure need not be limited to thedisclosed embodiments. It is intended to cover various modifications andsimilar arrangements included within the spirit and scope of the claims,the scope of which should be accorded the broadest interpretation so asto encompass all such modifications and similar structures. The presentdisclosure includes any and all embodiments of the following claims.

1. A method for reducing body weight in a mammal comprising: a.providing a composition containing an effective amount of purified andisolated3-(4-hydroxybenzylidene)-2-(2,5-dihydroxyohenyI)-1-(4-hydroxyphenyl)indane-4,6-diol;b. administering the composition to a mammal in need thereof; whereinthe effective amount of the composition is 10 mg to 900 mg daily.
 2. Amethod of claim 1, wherein the effective amount of the compositionprovided to the mammal is 50 mg to 200 mg daily.